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Growth method for natural killer cells

A technology of natural killer cells and lymphocytes, applied in animal cells, hybrid cell preparation, vertebrate cells, etc., can solve problems such as lack of security and failure to provide proliferation methods.

Active Publication Date: 2011-06-29
GREEN CROSS LAB CELL CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above mentioned only found new proliferation substances through the variants and evolved forms of IL-2 that have been used all the time, but did not provide a breakthrough proliferation method
[0008] Moreover, there are also reports about some researchers using tumor cell lines as feeder cells to proliferate NK cells, but most of them use tumor cell lines as feeder cells, etc. These methods are very important for clinical application. sex is not guaranteed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of feeder cells and isolation of natural killer cells (NK cells)

[0055] (1) Preparation of feeder cells

[0056] Take 20ml of peripheral blood from a healthy person, and put 5ml of the collected blood into a conical tube with a volume of 15ml. Add 5ml of normal saline to the blood and stir evenly with a pipette. Add 5ml of lymphocyte separation solution (ficoll) (GE healthcare, Uppsala, 17-1440-03) to a new 15ml conical tube with a volume capacity, and carefully add After the above-mentioned stirred (diluted) blood was centrifuged at 2000 rpm at room temperature for 30 minutes (Hanil Group, Korea, Union32-R).

[0057] After taking out the immune cell layer formed between the lymphocyte separation solution and plasma and moving it to a new 15ml conical tube, add HBSS to 10ml, mix the cells evenly, and then centrifuge at 1200rpm for 10 minutes. The supernatant was removed by vacuum suction. Then add 10ml of HBSS and repeat the centrifugation p...

Embodiment 2

[0066] Example 2: Culture of isolated natural killer cells

[0067] Nascent cells were cultured in 12-well plates (Falcon). Put 500ul of the feeder cells prepared in Example 1-(1) into the wells, and add 500ul of the natural killer cells separated in Example 1-(2) into the wells containing the feeder cells.

[0068] After adding IL-2 (Norvatis) cytokine at a concentration of 500 U / ml and OKT-3 antibody (ebioscience, 16-0037) at a concentration of 500 U / ml to each well containing cells, shake the well plate carefully to make the cells Mix well with cytokines.

[0069] The orifice plate was placed in a humid incubator at 37° C. containing 5% carbon dioxide for 5 days of culture, at this time, no culture medium or cytokines were added.

[0070] Counting from the culture day to day 5, pipette all the cells in the wells containing the cells into a 15 ml conical tube. 1 ml of cell culture solution was added to each well from which the cells were removed, and the remaining cells w...

Embodiment 3

[0076] Example 3: Analysis of the surface morphology of the obtained NK-cells

[0077] After removing OKT-3, only IL-2 was treated alone. On the 7th and 10th day, a part of the cells were harvested for surface morphology analysis.

[0078] Collect the cells before, during, or after the culture, centrifuge at 12000 rpm for 5 minutes, and remove the culture solution by vacuum suction. After diluting with 1 ml of FACS buffer (2.5% FBS+PBS), the number of cells was determined and diluted to 5×10 with FACS buffer. 6 cells / ml. 100 ul of the diluted cell solution was added to a FACS tube (Falcon), and antibodies were added as follows.

[0079] Test tube 1: no staining

[0080] Tube 2: anti-human CD3-FITC (BD Pharmingen, 5555339) + anti-human CD56-APC (BD Pharmingen, 555518) + anti-human CD16-PE (BD Pharmingen, 555407)

[0081] Tube 3: Anti-CD16-FITC (color control) (BD Pharmingen, 555406)

[0082] Tube 4: Anti-CD56-PE (color control) (BD Pharmingen, 555516)

[0083] Tube 5: ant...

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Abstract

The present invention relates to an improved growth method for natural killer cells (NK cells). More specifically, the present invention relates to a growth method for natural killer cells, which comprises a step of culturing natural killer cells in a medium containing anti-CD3 antibodies and interleukin proteins in the presence of peripheral blood leukocytes. The present invention provides an innovative growth method for natural killer cells, which can obtain a large amount of natural killer cells by remarkably increasing the growth rate compared with conventional growth methods for natural killer cells.

Description

technical field [0001] The invention relates to a proliferation method for obtaining high-yield natural killer cells (Natural Killer cells, NK cells). It further relates to a natural killer cell proliferation method comprising the step of culturing natural killer cells in a medium containing anti-CD3 antibody and interleukin protein in the presence of peripheral blood lymphocytes. Background technique [0002] Natural killer cells (hereinafter sometimes referred to simply as "NK cells") are cells of the lymphocyte lineage that play a role in immune responses. The cells have multiple functions, especially have a strong activity of killing tumor cells, so they are considered to be an important member of the immune surveillance system for eliminating tumorized or tumorizing abnormal cells in vivo. Therefore, researches on the effective use of the cells for tumor therapy or for the removal of virus-infected cells considered to be the origin of tumor development have long been i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/515C12N2501/23C12N5/06C12N5/0602C12N15/02
Inventor 安龙云郑美英许大锡黄琉炅
Owner GREEN CROSS LAB CELL CORP
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